In analysis methods using microscope-like arrangements it is known to analyze the luminescence lifetime, in particular the fluorescence lifetime, of certain materials, so-called fluorophores, so as to gain information on the analyzed object, e.g. the closer chemical environment of a fluorophore. In the field of microscopy this technique is typically referred to as FLIM (Fluorescence Lifetime Imaging Microscopy). Imaging measurements of fluorescence lifetime are further used in the medical field, e.g. for diagnosis on the eye, on the skin or on other organs. By way of example, fundus cameras are employed for medical analysis on the eye. Even if the mentioned medical analysis devices may significantly differ in their form from conventional microscopes they may be generally referred to as microscope-like optical devices.
FLIM measurements are typically performed as an alternative or in addition to intensity-based fluorescence measurements, because additional information concerning the fluorophores and their chemical environment can be gathered therefrom. By way of example, FLIM measurements may be advantageously employed if excitation and emission spectra of different fluorescent dyes differ only slightly. In this case, use can be made of the fact that fluorescent dyes having very similar spectra may have significantly different fluorescence lifetimes. Further, it is possible that unspecific auto-fluorescence signals of the investigated material are superimposed onto the fluorescence signals to be investigated. However, because auto-fluorescence is in most cases, associated with a significantly shorter fluorescence lifetime than, for example, the fluorescence lifetimes of purposefully employed fluorescent dyes, by means of FLIM measurements a distinction between auto-fluorescence, e.g., of a tissue sample, and the fluorescence from fluorescent dyes introduced into the tissue sample can be made. Further, it is possible to investigate auto-fluorescence of biological tissue samples themselves, e.g., to distinguish between healthy and pathologically modified tissue regions.
Further, FLIM measurements can be employed in connection with FRET experiments (FRET: Fluorescence Resonance Energy Transfer), in which energy of an excited molecule can be transferred to another molecule.
In measuring fluorescence lifetimes it is known to use measurement methods working in the time domain, i.e. so-called time-domain methods, or methods working in the frequency domain, i.e. so-called frequency-domain methods.
In the time-domain methods, typically a pulsed laser is used as a source of excitation radiation, which emits pulses in the range of femtoseconds to picoseconds. Then, the fluorescence decay curve is measured on a certain position of the sample. For this purpose, the sample is irradiated with laser light over a period of time which covers a larger number of laser pulses. The fluorescence response of the sample can then be analyzed e.g. using a method on the basis of time-correlated single photon counting. In this case, the time between excitation pulse and detecting the fluorescence photon is measured for each fluorescence photon. With a large number of detected fluorescence photons, a histogram is thereby obtained, which directly represents the fluorescence decay curve. This method can be used e.g. in connection with a confocal laser scanning microscope, in which the sample is scanned for imaging. However, it is also possible to use the method in connection with a wide-field illumination and to use a time-window controlled detection, a so-called “time-gated detection”. In this case e.g. a micro-channel plate may be arranged in front of a CCD-array (CCD: Charge Coupled Device). The amplification features of the micro-channel plate can be controlled by temporal variation of amplification voltages in the nanosecond range. In this way it is possible to define time windows for detecting the fluorescence radiation. By using two different time windows it is possible to analyze a mono-exponential decay process. However, the technical implementation of a time-domain method is typically technically complex and cost-intensive.
In the frequency-domain methods, the excitation radiation is periodically modulated, the modulation mostly being sinusoidal with frequencies from the kilohertz range to the gigahertz range. For generating the excitation radiation, e.g. an electro-optical modulator or an acousto-optical modulator in connection with a CW laser (CW: continuous wave) may be used. Alternatively, a laser which is pulsed in the picosecond range may be used.
Both mentioned method types for measuring fluorescence lifetimes are described in more detail in “Handbook of Biological Confocal Microscopy”, 2nd edition 1995, edited by James B. Poley, Plenum Press.
In European Patent Application, EP 1 162 827 A2, a measurement configuration and a method are proposed, in which the detection process of a CCD sensor is influenced to make possible a phase-sensitive measurement. For generating the excitation radiation, it is proposed to use a blue emitting LED (LED: Light Emitting Diode), which is modulated with frequencies in the kilohertz range. However, the used CCD sensor requires specific modifications, which imply a high outlay and high costs for a practical application in microscopy or medical diagnostics.
In US 2007/0057198 A1, a method for measuring fluorescence lifetimes is described, in which the intensity of the excitation radiation is periodically changed. The method is preferably used in connection with a laser-scanning microscope.
In Schwarte, R., “Dynamic 3D-Vision”, International Symposium on Electron Devices for Microwave and Optoelectronic Applications 2001, Vienna, Austria, 15-16 Nov. 2001, the operation of a time-of-flight camera is described, which is referred to as a photonic mixer device (PMD). It is mentioned that such time-of-flight cameras may also be used in the framework of FLIM measurements.
European Patent Application, EP 1 746 410 A1, proposes using a so-called “lock-in imager” in FLIM measurements. The operation of this lock-in imager substantially corresponds to that of a time-of-flight camera. In particular, it is proposed to use a microscope configuration with a dark-field illumination. The excitation radiation is generated by a laser diode or LED.
However, in the known methods for determining fluorescence lifetimes, there are problems in that, in an object under investigation, there are often multiple sources of fluorescence radiation, the fluorescence responses of which are superimposed onto each other. Typically, it is, therefore, merely possible to determine an average fluorescence lifetime of all involved fluorophores.